Piotr Bednarczyk

Hypoxia increases activity of the BK-channel in the inner mitochondrial membrane and reduces activity of the permeability transition pore.

Hypoxia can cause severe damage to cells by initiating signaling cascades that lead to cell death. A cellular oxygen sensor, other than the respiratory chain, might exist in sensitive components of these signaling cascades. Recently, we found evidence that mitochondrial ion channels are sensitive to low levels of oxygen. We therefore studied the effects of hypoxia on the mitochondrial BK-channel (mtBK), on the mitochondrial permeability transition pore (PTP), and on their possible interaction. Using single-channel patch-clamp techniques we found that hypoxia inhibited the PTP but substantially increased the mtBK activity of mitoplasts from rat liver and astrocytes. Experiments measuring the mitochondrial membrane potential of intact rat brain mitochondria (using the fluorescence dye safranine O) during hypoxia exhibited an increased Ca2+-retention capacity implying an impaired opening of the PTP. We also found a reduced Ca2+-retention capacity with 100 nM iberiotoxin, a selective inhibitor of BK-channels. We therefore conclude that there is interaction between the mtBK and the PTP in a way that an open mtBK keeps the PTP closed. Thus, the response of mitochondrial ion channels to hypoxia could be interpreted as anti-apoptotic.

Calcium Ions Regulate K + Uptake into Brain Mitochondria: The Evidence for a Novel Potassium Channel

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca2+ additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca2+ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BKCa channel). Furthermore, NS1619 – a BKCa channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca2+. These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-β4 subunit (of the BKCa channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of β4 subunit in neuronal mitochondria. We hypothesize that the mitochondrial BKCa channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.

A novel potassium channel in skeletal muscle mitochondria

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

Pharmacology of mitochondrial potassium channels: dark side of the field

Mitochondrial potassium channels play an important role in cytoprotection. Potassium channels in the inner mitochondrial membrane are modulated by inhibitors and activators (potassium channel openers) previously described for plasma membrane potassium channels. The majority of mitochondrial potassium channel modulators exhibit a broad spectrum of off‐target effects. These include uncoupling properties, inhibition of the respiratory chain and effects on cellular calcium homeostasis. Therefore, the rational application of channel inhibitors or activators is crucial to understanding the cellular consequences of mitochondrial channel inhibition or activation. Moreover, understanding their side‐effects should facilitate the design of a specific mitochondrial channel opener with cytoprotective properties. In this review, we discuss the complex interactions of potassium channel inhibitors and activators with cellular structures.

Determination of the rate of K + movement through potassium channels in isolated rat heart and liver mitochondria

Both ATP-regulated (mitoK(ATP)) and large conductance calcium-activated (mitoBK(Ca)) potassium channels have been proposed to regulate mitochondrial K(+) influx and matrix volume and to mediate cardiac ischaemic preconditioning (IP). However, the specificity of the pharmacological agents used in these studies and the mechanisms underlying their effects on IP remain controversial. Here we used increasing concentrations of K(+)-ionophore (valinomycin) to stimulate respiration by rat liver and heart mitochondria in the presence of the K(+)/H(+) exchanger nigericin. This allowed rates of valinomycin-induced K(+) influx to be determined whilst parallel measurements of light scattering (A(520)) and matrix volume ((3)H(2)O and [(14)C]-sucrose) enabled rates of K(+) influx to be correlated with increases in matrix volume. Light scattering readily detected an increase in K(+) influx of <5 nmol K(+) min(-1) per mg protein corresponding to <2% mitochondrial matrix volume increase. In agreement with earlier data no light-scattering changes were observed in response to any mitoK(ATP) channel openers or blockers. However, the mitoBK(Ca) opener NS1619 (10-50 microM) did decrease light scattering slightly, but this was also seen in K(+)-free medium and was accompanied by uncoupling. Contrary to prediction, the mitoBK(Ca) blocker paxilline (10-50 microM) decreased rather than increased light scattering, and it also slightly uncoupled respiration. Our data argue against the presence of significant activities of either the mitoK(ATP) or the mitoBK(Ca) channel in rat liver and heart mitochondria and provide further evidence that preconditioning induced by pharmacological openers of these channels is more likely to involve alternative mechanisms.

Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart

Mitochondrial ATP‐regulated potassium (mitoKATP) channels play an important role in cardioprotection. Single channel activity was measured after reconstitution of inner mitochondrial membranes from bovine myocardium into a planar lipid bilayer. After incorporation, the potassium channel was recorded with a mean conductance of 103 ± 9 pS. The channel activity was inhibited by ATP/Mg and activated by GDP. Magnesium ions alone affected, in a dose dependent manner, both the channel conductance and the open probability. Magnesium ions regulated the mitoKATP channel only when added to the trans compartment. We conclude that Mg2+ regulates the cardiac mitoKATP channel from the matrix site by affecting both the channel conductance and gating.

ATP-sensitive potassium channel in mitochondria of the eukaryotic microorganism Acanthamoeba castellanii.

We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP+-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mm ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 ± 7to166 ± 10 picosiemens, inhibition by 1 mm ATP/Mg2+ or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria.

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain.

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca2+ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109+/-6pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg(2+) complex and Ca(2+) ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.

Large-conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells

In the present study, we describe the existence of a large-conductance Ca²⁺-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca²⁺, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β₂-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.